Suspended ultra-sound microbubble imaging

ABSTRACT

An ultrasonic imaging technique is disclosed which uses microbubbles as echo contrast agents. In general the method employs maintenance of an ultrasound signal while the contrast agent is intravenously injected into a mammal. Once all the contrast agent has been injected and transmission of the signal is suspended for a period of time sufficient for the microbubbles perfuse the organ of interest. Transmission of the ultrasound signal is then resumed and peak contrast images are obtained which rival more complicated imaging procedures such as nuclear resonance imaging.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of co-pending U.S. patent application entitled SUSPENDED ULTRA-SOUND INDUCED MICROBUBBLE CAVITATION IMAGING, Ser. No. 08/439,619, filed May 12, 1995 by Thomas R. Porter.

BACKGROUND OF THE INVENTION

Ultrasonic imaging is used as a diagnostic tool to aid in therapeutic procedures. It is based on the principle that waves of sound energy can be focused upon an area of interest and reflected to produce an image. Generally, an ultrasonic transducer is placed on a body surface overlying the area to be imaged, and ultrasonic energy, produced by generating and receiving sound waves is transmitted. The ultrasonic energy is reflected back to the transducer where it is translated into an ultrasonic image. The amount and characteristics of the reflected energy depend upon the acoustic properties of the tissues, and contrast agents which are echogenic are preferentially used to create ultrasonic energy in an area of interest and improve the image received.

In ultrasound imaging, videotape images obtained following contrast injection are digitized, allowing the gray scale to be quantified from 1 to 225 gray scale units for 30 cardiac cycles. The contrast intensity is plotted on the vertical axis against time on the horizontal axis. The peak videointensity (corrected for baseline intensity) is determined as the highest point on the time intensity curve.

For a discussion of contrast echographic instrumentation, see, for example, De Jong N, "Acoustic properties of ultrasound contrast agents", CIP-GEGEVENS KONINKLIJKE BIBLIOTHEEK, DEN HAG (1993), pages 120 et seq.

Contrast echocardiography has been used to delineate intracardiac structures, assess valvular competence, and demonstrate intracardiac shunts. Myocardial contrast echocardiography (MCE) has been used to measure coronary blood flow reserve in humans. MCE has been found to be a safe and useful technique for evaluating relative changes in myocardial perfusion and delineating areas at risk.

A multiplicity of potential ultrasonic imaging agents has been reported for contrast echocardiography. No such agent routinely attains visually discernible myocardial uptake following peripheral intravenous injection. Although there have been many reports of transpulmonary transmission of ultrasound contrast agents following intravenous injection and despite the fact that myocardial opacification on echocardiogram can be produced by left sided injection of such contrast agents, visualization of myocardial contrast has not been achieved by intravenous administration of sonicated microbubbles.

Most recently, sonicated albumin and sonicated dextrose/albumin have been shown to produce variable degrees of left ventricular chamber ultrasound contrast following intravenous injection. (See Villanueva et al. Circulation 85:1557-1564, 1992; Lin et al. Int J Card Imaging 8:53-6, 1992; Feinstein et al. J Am Coll Cardiol 16:316-224, 1990; Keller et al. Am Heart J 114:570-575, 1987; and Shapiro et al. J Am Coll Cardiol 16:1603-1607, 1990). The microbubbles of these contrast agents are small (4-6 microns) and are capable of swift transpulmonary passage. However, visually discernible myocardial uptake of such microbubbles following peripheral intravenous injection has not been possible because of the rapid diffusion of blood soluble oxygen and nitrogen inside the microbubble into the blood which consequently loses its ultrasound reflective properties (e.g., see Porter et al. J Am Soc Echocard Supplement 7:S1, May 1994, and Weyman AE: "Principles and Practice of Echocardiography", Malvern, Pennsylvania: Lea & Febiger, 1994; pp. 302-26.)

Despite recent advances in contrast agents comprising injectable gas encapsulated microbubbles, several problems remain for adequate detection and visualization of the organ of interest. The bubbles are plagued with filtration by capillaries, diffusion of gas to the liquid medium, and lack of concentration at the organ of interest due to dilution.

Attempts to solve these problems have led to studies of acoustic velocity of media containing gas bubbles, second harmonic emission, and resonance frequency. These studies to date have met with little improvement in contrast visualization.

It is an object of the present invention to provide a safe, simple and effective method to visualize and improve an ultrasound image following injection of gas-filled microbubbles.

Other objects of the invention will be apparent from the description of the invention which follows.

SUMMARY OF THE INVENTION

According to the invention, after introduction of a microbubble contrast agent the ultrasound transmission is suspended and is then resumed for a striking improvement in contrast. It is postulated that suspension of the ultrasound relieves pressure on the microbubbles allowing them to expand or to increase their longevity before diffusing into the blood. Use of this method results in up to ten times improvement in contrast, decreases acoustic shadowing and allows for reduction in the amount of contrast agent necessary, thereby reducing toxicity and improving safety.

DETAILED DESCRIPTION OF THE INVENTION

The primary problem in contrast ultrasound imaging is the detection and quantification of perfusion in the anatomical organs of interest, such as heart, kidney, liver etc. The most success to date has been with using contrast gas-filled microbubbles which are injected intravenously into the bloodstream.

Often, however, the intravenously injected bolus of microbubbles gets diluted in concentration as it travels to the targeted organ of interest, making detection difficult if not impossible. One of the primary problems in echocardiography is the detection of myocardial perfusion. More importantly, as the bolus flows through the right-side of the heart and passes through the lungs, microbubbles greater than 8 microns in size are filtered out by the pulmonary capillaries.

Further, as the bubbles flow into the left-side of the heart, some of the bubbles are destroyed during the course of the heart cycle as they are subjected to the fairly high systolic pressures in the left ventricle (LV). Some of the bubbles disappear by the simple process of diffusion of gas into the surrounding liquid medium.

Finally, the bubbles in the ventricle strongly attenuate the incident ultrasound field and cast an acoustic shadow on the distal myocardium. Hence, there is a trade-off between increasing the dose for improved microbubble detection sensitivity and the need for minimizing the acoustical shadowing of the myocardial beds. All of this is exacerbated by the fact that only about 4% of the total blood volume in the LV cavity enters the coronary circulation. As a net result, only a small number of microbubbles of small size (from the original bolus injection) traverse the myocardial vasculature.

This invention alleviates these concerns by allowing for detection of very small amounts of contrast agent, improving contrast by as much as ten times and can be used in echocardiography as well as for renal or hepatic imaging. The invention (suspended ultrasound imaging) comprises a modification of standard ultrasound imaging techniques and can be used with any ultrasound imaging device in combination with a microbubble contrast agent.

In this method the ultrasound signal is suspended and re-initiated (i.e. the transducer is held in position over the organ of interest, for example the heart) upon introduction of the contrast agent. The contrast agent is injected peripherally, in an amount sufficient for visual detection of the organ of interest. Standard amounts of microbubble contrast agent may be used, from about 0.04 ml/kg to about 0.08 ml/kg for humans however it will be seen that with the practice of this invention much smaller amounts will work equally as well, as little as 0.001 ml/kg to 0.0025 ml/kg. The dose range is patient specific as large patients may require slightly higher doses to produce equivalent left ventricular contrast. Standard methodology for contrast echocardiography are described in Weyman, Arthur E., "Principles and Practice of Echocardiography" Lea and Fibiger, Malvern, Pa. (1994 2d Ed).

After injection the transmission of the ultrasound signal is interrupted, or suspended. In one embodiment the suspension is for a period of time sufficient for the contrast agent to perfuse the organ of interest. The time period will generally vary according to the organ of interest. It must only be long enough for some of the agent to have reached the organ of interest. For the heart this would be the time sufficient for the contrast agent to reach the myocardium. This is approximately about 10-120 seconds, preferably around 20-30 seconds and should not vary significantly from patient to patient.

In another embodiment, the ultrasound signal can be suspended and re-initiated in a pulsing manner, (i.e. in intervals of approximately one cardiac cycle or more). For example, the ultrasound transmission or pulses can be triggered or gated to only one part of the cardiac cycle. This triggered or gated event consists of anywhere from 1 to 150 or more cycles or periods of ultrasound waves or pulses, depending on how the ultrasound signal is calibrated by the manufacturer. This can allow for transient imaging from injection until dissolution of the contrast agent. The number of the pulses in each triggered or gated event is dependent on the frequency as well as the number of wavelengths where t=1/f, where t is the time in seconds for one wavelength. For 2.5 MHz frequency the length of one pulse would be 0.4 microseconds. As noted above, the number of pulses within each triggered or gated event is variable and ranges from 1 to 150 or more wavelengths. For the rest of the interval between triggered or gated events there is no energy imparted to the animal. In a preferred embodiment the method is used with second harmonic imaging where the signal is received at a frequency which is a harmonic interval of the transmission signal. The process and concept of secondary harmonic imaging is disclosed in Uhlendorf et al in U.S. Pat. No. 5,410,516 ULTRASOUND PROCESSES AND CIRCUITS FOR PERFORMING THEM which is incorporated herein by reference. According to the invention a much lower ultrasound transmitting frequency can be used, decreasing attenuation. Transmission frequencies as low as 1.8 MHz have been efficiently employed with the method of the invention and could likely go much lower.

Upon resumption of the signal the image is then detected either at the transmitting frequency or at a harmonic thereof.

In echocardiography there will be a uniform opacification of the entire myocardium. Within a few seconds, the intensity of the entire myocardium uniformly drops to the original (dark) intensity level. Regions of the myocardial bed that are devoid of blood flow and contrast remain dark at all times. Hence, it is easy to differentiate between normally perfused and abnormally perfused myocardial regions.

While not wishing to be bound by any theory, it is postulated that the observed phenomenon arises as a result of the transient response of the contrast microbubbles when subjected to a driving ultrasonic field. The ultrasound signal causes the microbubbles to compress, or cavitate. When the microbubbles are injected, they become compressed, as they encounter the ultrasonic field. When the ultrasound signal is suspended, the transmit power into the anatomy is shut off. In the absence of a radiating field the pressure on the microbubbles will be reduced, the microbubbles in the myocardium or other organ, grow in size by a finite amount and the longevity of the bubbles is increased. Some may even coalesce to form larger scatterers.

The reflection of the ultrasound signal will then be enhanced by the larger size of the microbubbles. This is because the backscattered ultrasound energy is directly proportional to the number of scatterers in the region of interest and varies as the sixth power of the radius of the scatterer.

When the ultrasound system is resumed, the transmit power into the anatomy is enabled and will generate the image scan on the display. Peak contrast images are obtained at this point, before the ultrasound signal begins to cavitate the microbubbles once again. As the ultrasound field builds up from zero to steady state, the pressure on the microbubbles increases causing them to diminish in size once again. This transient decrease in scatterer size causes the resultant transient decrease in myocardial intensity.

It is postulated that another explanation for the increase in intensity upon resuming ultrasound transmission may be that the impressed ultrasound field sputters the microbubbles and any coalesced microbubble aggregates into numerous microbubbles thereby effectively increasing the number of scatterers and thereby causing the increased myocardial opacification. The contrast is so improved that the image produced rivals that of more complicated imaging such as nuclear resonance imaging.

The main advantage of this invention is that it allows for sensitive detection and quantification of perfusion at the target organ in a non-invasive mode using a very small dose of contrast microbubbles without producing acoustical shadows. Because of the small dose, patient safety is enhanced and the cost is also minimized.

For most ultrasound imaging, the contrast agent is formulated in a pharmaceutically effective dosage form for peripheral administration to the host to be imaged. Generally such host is a human subject although other mammalian hosts, such as canine or equine can be imaged effectively. In a most preferred embodiment the contrast agent is a sonicated mixture of commercially available albumin (human), USP, solution (generally supplied as 5% or a 25%, by weight, sterile aqueous solutions), and commercially available dextrose, USP, for intravenous administration are employed. This mixture is sonicated under ambient conditions, i.e., room air, temperature and pressure, and is perfused with perfluorocarbon or other commercially available inert gas (99.9% by weight) during sonication.

In a preferred embodiment the invention uses a microbubble contrast agent wherein the microbubbles are stabilized by a filmogenic, de-naturable protein coating. Suitable proteins include naturally occurring proteins such as albumin, human gamma-globulin, human apotransferrin, Beta-lactose, and urease. The invention preferably employs a naturally occurring protein, but synthetic proteins may also be used. Particularly preferred is human serum albumin.

Although intravenous echo contrast agents made from sonicated microbubbles are known (e.g., ALBUNEX, Molecular Biosystems, Inc.) and can be employed in this invention, it is preferred to use a sonicated aqueous solution containing a mixture of a pharmaceutically acceptable saccharide, e.g., dextrose, and a protein, e.g., albumin. Generally, sonication is performed in an air atmosphere. In an especially preferred embodiment, dextrose, which is readily available in pharmaceutically acceptable dosage forms, is the preferred saccharide and human serum albumin is the preferred protein. The preferred embodiment would include a two-fold to eight-fold dilution of 5%-50% by weight of dextrose and a 2%-10% by weight of human serum albumin. Exemplary of other saccharide solutions of this invention are an aqueous monosaccharide solution (e.g. having the formula C₆ H₆ O₁₂, such as, the hexoses, dextrose or fructose, or mixtures thereof), aqueous disaccharide solution (e.g., having the formula C₁₂ H₂₂ O₁₁, such as sucrose, lactose or maltose, or mixture thereof), or aqueous polysaccharide solution (e.g., soluble starches having the formula (C₆ H₁₀ O₅)_(n), wherein n is a whole integer between about 20 and about 200, such as amylase or dextran, or mixtures thereof. Sonication by ultrasonic energy causes cavitation within the dextrose-albumin solution at sites of particulate matter or gas in the fluid. These cavitation sites eventually resonate and produce small microbubbles (about 4 to about 7 microns in size) which are non-collapsing and stable. In general, sonication conditions which produce concentrations of greater than about 4×10⁸ m of between about 5 and about 6 micron microbubbles are preferred.

The mean microbubble size of sonicated dextrose albumin ranges from between about 5 to about 6 microns. This is a good size as it has been observed that microbubble radius decreases as a function of time in a still liquid due to a diffusion gradient present between the internal and external gases of the microbubble. An increase in microbubble size has a significant effect on the persistence of a microbubble within blood. It must also be of a size sufficient for transpulmonary passage. It must have a mean diameter of less than 10 microns and greater 0.1 microns. Since the size of albumin microbubbles is ideal (between 5 and 6 microns) for transpulmonary passage, the major reason for the significant loss in left ventricular and myocardial videointensity produced following intravenous injection of albumin coated microbubbles is due to the significant diffusion of gases within the microbubble following intravenous injection during transit to the left ventricular cavity. Sonicated dextrose albumin enhanced with an inert gas such as perfluorocarbon gas, having a lower blood solubility than air, and a molecular weight of greater than 100 grams/mole, produces a significantly higher left ventricular and myocardial videointensity than sonicated albumin alone.

Because of high surface tension, the concentration of nitrogen and oxygen gas within the microbubble is much higher than that in blood, and thus there is a rapid diffusion of this gas into the blood stream following intravenous injection. Wible et al. (Circulation, 88:I-401, 1993) have demonstrated that this diffusion process can be accelerated if one decreased the partial pressure of nitrogen within the blood stream by decreasing the inhaled fraction of nitrogen. This lower external concentration of nitrogen results in loss of the left ventricular videointensity produced by the same intravenous injection of sonicated albumin while inhaling room air. It has also been observed that oxygen rapidly diffuses out of gas bubbles into human blood (See Yang et al., J Biomech 3:275, 1971).

Both nitrogen and oxygen diffuse rapidly across these concentration gradients, but nitrogen appears to dissolve more slowly than oxygen into blood. Since nitrogen is the major component of air, decreasing the concentration gradient for nitrogen across the microbubble improves left ventricular and myocardial videointensity following intravenous injection. Exposing the microbubbles to a non-toxic gas having a lower blood solubility and/or microbubble diffusivity than that of nitrogen and having a gas density of greater than about 0.300 lb/ft³ during sonication increases the size and stability of the microbubbles in sonicated dextrose albumin, while lowering the solubility and diffusivity of the microbubbles in blood. Suitable gases are those which are gas at 37° C. and which are nontoxic. Insoluble gases useful for contrast agents include but are not limited to prefluorocarbon gases such as perfluoromethane, perfluoroethane, perfluoropropane, perflouorobutane, perfluoropentane etc, or sulfur hexafluoride. In a preferred embodiment the gas is perfluoropropane (C₃ F₈) or perfluorobutane (C₄ F₁₀). The perfluorocarbon gas content of the microbubbles is sufficient to lower microbubble gas solubility and diffusivity in vivo in blood. Generally, the minimum amount of insoluble gas in the microbubbles which is effective is that amount which results in microbubbles which pass reliably through the pulmonary circulation without collapse. This is evidenced by opacification of the myocardium of the left ventricle of the heart following intravenous injection and can be visually discerned by echocardiography.

In addition to myocardial imaging the contrast agents of this invention are useful for renal and hepatic imaging. Thus, another embodiment of this invention provides a method for myocardial, renal or hepatic opacification. The method preferred involves obtaining an echo contrast agent of this invention, introducing said echo contrast agent into a host by intravenous injection, and performing an echo contrast study on said host using a suitable Doppler or ultrasound echo apparatus as discussed more fully hereinafter.

In a most preferred embodiment the contrast agent is a perfluorobutane-enhanced sonicated dextrose albumin solution comprised of a sonicated three-fold dilution of 5% human serum albumin with 5% dextrose. During sonication, said solution is perfused with perfluoropropane for about 80 seconds, and concomitantly exposed to perfluorobutane or perfluoropropane for at least about 5 seconds during sonication. This lowers the solubility and diffusivity of the microbubble gas. The resulting microbubbles are concentrated at room temperature for at least about 120±5 minutes, wherein the excess solution settles in the sonicating syringe. The excess solution is expelled and the concentrated microbubbles are transferred to a sterile syringe and injected intravenously into a mammal.

Using the method of the invention in echocardiography will result in a higher degree of myocardial opacification, endocardial border delineation, and enhanced detection of left-sided ultrasound Doppler signals, upon peripheral venous administration. Additionally, the method of the invention allow for increased sensitivity as small doses of microbubbles are easily detected, which subsequently enables ultrasonic visualization of the liver and kidneys following an intravenous injection.

The following examples are for illustration purposes only and are in no way intended to limit the invention. It will be apparent to those of skill in the art that other embodiments are practicable and even intended.

EXAMPLE 1 Preparation of Contrast Agents

Albumin (human) USP, 5% solution (hereinafter referred to as "albumin") and dextrose USP, 5% solution (hereinafter referred to as "dextrose") were obtained from a commercial source. The sonicating system used for sonication was a Heat System Ultrasonic Processor Model XL2020 (Heat Systems Inc., Farmingdale, N.Y.). The 1/2 inch horn transducer was a resonating piezoelectric device. The 1/2 inch sonicating horn tip was sterilized prior to each sonication.

Sonication of Samples

Sixteen milliliter aliquots of albumin diluted 1:3 with dextrose were drawn up into a 35 cc "Monoject" syringe (Becton Dickinson and Company, Rutherford, N.J.) and sonicated for 80±1 seconds. The "Leur-Lok" of the 35 milliliter syringe is then attached to a stopcock. After mixing the dextrose albumin solution by hand for about 7 to about 10 seconds, the plunger was removed from the top of the syringe. The sterile sonicating horn was then lowered into the open end of the syringe until at the surface of the albumin-dextrose solution. The solution was placed at the horn tip and manually held at this position while continuously sonicating at a frequency of 20,000 Hz and a power output of 210 W for 80±1 seconds to form a stable microbubble solution.

Gas Perfusion of Samples

During sonication, the dextrose albumin mixture was exposed to either perfluorobutane or perfluoropropane gas (Commercial Grade, 99.9% by weight). The gas was drawn up into a sterile syringe through a 0.22 μM filter (Micron Separations Inc., Westborough, Mass.) to prevent contamination. During sonication, 5 milliliters of the perfluorocarbon gas was manually injected into the solution, over the 80 second time interval, through the stopcock so that the microbubbles produced contained this less soluble gas. The total volume of perfluorobutane-enhanced sonicated dextrose albumin (BESDA) or perfluoropropane-enhanced sonicated dextrose albumin (PESDA) produced with this formulation was 25±2 milliliters. These samples were then used for intravenous injection.

Microbubble Analysis

Microbubble size and purity was determined using hemocytometry. Microscopic inspection of the microbubbles was performed to determine if any coalescent microbubbles were present in the solution. Microbubble concentration was determined using a Coulter Counter. The contrast agent was rejected for use if any of the following conditions were present: the mean microbubble size is 4.0 to 6.0 microns; coalesced microbubbles or strands were detected by light microscopy; or the mean microbubble concentration was less than 0.8×10⁹ or greater than 1.5×10⁹ microbubble/milliliter. The sample was also rejected if the number of microbubbles greater than 10 microns in the sample was greater than 4%.

All samples were stored in 35 milliliter syringes until time of injection. All solutions were given within 36 hours of production. All samples were prepared in a laminar flow hood.

EXAMPLE 2 Preparation of Open-Chest Dogs

Mongrel dogs of either sex (15-30 kilograms) were anesthetized with sodium pentobarbital (30 milligram per kilogram intravenously), incubated, and ventilated initially with room air using a positive pressure respirator. A left thoracotomy was performed under sterile conditions and the pericardium incised. In addition to a 19 gauge peripheral intravenous line, eight French Catheters were placed in the femoral artery and vein for intravenous administration of ultrasound contrast agents and pressure monitoring. Through one femoral venous sheath, a 7F balloon-tipped thermodilution catheter was placed in the pulmonary artery using fluoroscopy for determination of pulmonary artery pressure and cardiac output. A 7F pigtail catheter was introduced into the left ventricle for pressure measurements (left ventricular systolic and end-diastolic pressure) following injection of each ultrasound contrast agent.

Following adequate surgical exposure, a 3.5 Megahertz ultrasound transducer connected to a commercially available ultrasound scanner (Hewlett Packard Company; Andover, Mass.) was placed in a warm water bath. The bath overlays the anterior epicardial surface. The transducer was mounted on a clamp and lowered into the bath. It was adjusted until an optimal stable short axis view of the left and right ventricle had been obtained at the ventricular mid-papillary muscle level. These images were then used to assess left ventricular cavity and myocardial uptake of contrast following intravenous injection.

EXAMPLE 3 Delayed Ultrasound-Induced Cavitation Imaging vs. Conventional Ultrasound Imaging for Echocardiography

To determine if the acoustic shadowing could be decreased or eliminated with the method of the invention, four human patients were tested comparing conventional ultrasound imaging with delayed ultrasound-induced cavitation imaging. For this experiment, myocardial contrast was determined using on-line digitally acquired videointensity obtained from software supplied in conjunction with the commercially available ultrasound system (Hewlett-Packard Sonos 1500 Phased Array imaging system, Hewlett Packard, Andover, Mass.). The peak myocardial videointensity (PMVI), duration of acoustic shadowing (AS), and percent of myocardial contrast (MC) were observed. The results are shown in Table 1 below, and actual representative ultrasound images are shown in FIGS. 1(a)-(d).

Each patient was injected intravenously with 0.0025-0.005 milliliter per kilogram of contrast agent, prepared as in Example 1. The fluorocarbon gas agent used was decafluorobutane. For the conventional ultrasound imaging, the transmission of ultrasound was constant at 2.5-2.7 MHz and imaging was performed continuously throughout the experiment. Peak contrast images of the myocardium were obtained once the microbubbles of the contrast agent reached the myocardium.

For the delayed ultrasound-induced cavitation imaging, the transmission of ultrasound was maintained at 2.5-2.7 MHz until the patient was injected with contrast agent. Upon intravenous injection of contrast agent, the ultrasound transmission was suspended for 20 to 30 seconds. Ultrasound transmission was resumed at 2.5-2.7 MHz once the microbubbles of the contrast agent reached the myocardium. Peak myocardial contrast images were obtained immediately following commencement of the ultrasound transmission and before the microbubbles began to compress.

                  TABLE 1                                                          ______________________________________                                         Myocardial Contrast in Ultrasound Imaging                                                               Ultra-      Acous-                                                             sound       tic   Myo-                                                 Dose of Signal      Shad- cardial                             Imaging          Contrast                                                                               Delay       owing Contrast                            Modality                                                                              Patient # Agent   Time  PMVI  (AS)  (MC)                                ______________________________________                                         UIC*   1) 2.7 MHz                                                                               0.005   20    19    10 sec                                                                               2+                                         2) 2.5 MHz                                                                               0.0025  20    11    23 sec                                                                               2+                                         3) 2.5 MHz                                                                               0.0025  25    1      8 sec                                                                               1+                                         4) 2.5 MHz                                                                               0.005   30    14    12 sec                                                                               2+                                  CONV** 1) 2.7 MHz                                                                               0.005   0     5     20 sec                                                                               2+                                         2) 2.5 MHz                                                                               0.0025  0     0     25 sec                                                                               1+                                         3) 2.5 MHz                                                                               0.0025  0     3     20 sec                                                                               1+                                         4) 2.5 MHz                                                                               0.005   0     3     30 sec                                                                               2+                                  ______________________________________                                          *delayed ultrasound induced cavitation                                         **conventional ultrasound imaging                                        

The results of this experiment indicate that conventional imaging following intravenous injection of the perfluorocarbon-enhanced sonicated dextrose albumin contrast agent produced a high degree of acoustic shadowing in the posterior structures of the left ventricular cavity. The delayed ultrasound-induced cavitation imaging decreased the amount of acoustic shadowing by a sufficient amount to enable the imaging of inferior defects of the myocardium. The method of the invention was also compared to standard myocardial imaging in dogs with an increase in myocardial contrast of ten times that of conventional method. A marked reduction in acoustic shadowing was also seen.

EXAMPLE 4 Effects of Transducer Frequency and Power on Cavitation Ultrasound Imaging

The effects of transducer frequency and transducer acoustic output was demonstrated in the method of the invention using two open chest dogs.

Two dogs received a total of 22 intravenous injections of a perfluoropropane-enhanced sonicated dextrose albumin contrast agent (prepared as in Example 1) at doses between 0.005 and 0.010 milliliters per kilogram body weight. The diagnostic ultrasound signal was transmitted only after the contrast agent microbubbles had reached the myocardium and peak myocardial videointensity (PMVI) and the duration of myocardial contrast (dur-MC) were measured following each injection. The transducer frequency and acoustic output were varied between each injection. The results are shown in Table 2 below.

                  TABLE 2                                                          ______________________________________                                         Effects of Transducer Frequency and                                            Power on Cavitation Ultrasound Imaging                                         Transducer                    Duration of                                      Frequency                                                                              Acoustic Output       myocardial contrast                              (MHz)   (decibels)    PMVI    (dur-MC)                                         ______________________________________                                         2.0-2.5 158-170       42 ± 6                                                                              5 ± 1                                         2.0-2.5 212           43 ± 6                                                                               1 ± 1*                                       2.7-3.5 158-170       37 ± 7                                                                               11 ± 1**                                     2.7-3.5 212           31 ± 7                                                                              3 ± 1                                         ______________________________________                                          *p < 0.05, compared to other transducer frequencies                            **p < 0.05, compared to other durations                                  

The results indicate that the best myocardial contrast imaging is obtained when the transducer frequency is low and a low acoustic output prolongs the duration of this phenomenon.

EXAMPLE 5 Pulsed Ultrasound Imaging

Another example involved five mongrel dogs. Each dog was placed under general anesthesia with intravenous sodium pentobarbital, incubated, and placed on a respirator inspiring room air. An open lateral thoracotomy was performed and the heart suspended in a pericardial cradle. In one dog, however, imaging was performed prior to thoracotomy from the chest wall. A 2.0 millimeter transit time ultrasound coronary flow cuff (S-series; Transonics, Inc.; Ithaca, N.Y.) was placed around the left anterior descending and left circumflex coronary arteries to monitor coronary flow. A 7 French pulmonary artery catheter capable of measuring thermodilution cardiac output and a 7 French pigtail catheter were used to monitor pulmonary artery and left ventricular pressures.

Ultrasound images in the first two dogs were obtained using a 3.5 Megahertz ultrasound transducer connected to a commercially available scanner (Hewlett Packard. Sonos 1500; Andover, Mass.). In the last three dogs, a prototype 2.5 MHz transducer equipped to receive reflected and scattered frequencies in the second harmonic (5.0 MHz) was used in conjunction with a Hewlett-Packard Sonos 2500 imaging system. The transducer was placed in a warm water bath overlying the anterior epicardial surface of the heart, and adjusted to produce a short axis view of the left ventricle at the mid-papillary muscle level. A 2-0 silk ligature was placed around either the left anterior descending or left circumflex artery in three dogs in order to create a critical coronary narrowing which reduced resting coronary flow, or to create total vessel occlusion. The entire study was approved by the Institutional Animal Care and Use Committee and was in compliance with the Position of the American Heart Association on Research Animal use.

Preparation and Standardization of PESDA

Five percent human serum albumin and 5% dextrose were obtained from the hospital pharmacy. Three parts of 5% dextrose and one part 5% human serum albumin (total 16 milliliters) were drawn into a 35-milliliter Monoject syringe. Each dextrose albumin sample was hand agitated with 8±2 milliliters of a fluorocarbon gas (decafluorobutane, C₄ F₁₀ ; Molecular weight 238 grams/mole), and the sample was then exposed to electromechanical sonication for 80±5 seconds. The mean size of four consecutive samples of PESDA microbubbles produced in this matter measured with hemocytometry was 4.6±0.4 microns, and mean concentration measured by a Coulter counter was 1.4×10⁹ bubbles/milliliter.

In order to test how much better the transient contrast produced by brief or gated exposure to pulsed ultrasound (suspended ultrasound imaging) was than conventional imaging, the study protocol was performed in one of two ways. One method involved comparing the peak myocardial videointensity produced by conventional imaging with the transient myocardial contrast that occurred immediately after freezing transducer output for the first 10-20 seconds following intravenous injection of a low dose (0.005-0.01 milliliter per kilogram) of PESDA (Method 1). This time period was determined from a previous test injection which counted the time required for contrast to reach the left ventricular cavity and for acoustic shadowing in this cavity to resolve. Since the transient myocardial contrast appeared to be produced pulsed ultrasound was temporarily interrupted, a second method of enhancing this response was employed by delivering pulses gated (triggered) to only one part of the cardiac cycle (Method 2).

The non-linear properties of transient response imaging and its frequency dependence were also tested. The effect of transducer frequency on the magnitude of contrast produced was compared in two dogs by assessing differences in the contrast intensity using 2.5 versus 3.5 MHz imaging. In dogs 3 through 5, comparisons of anterior and posterior myocardial videointensity between CI and TRI were made with a prototype second harmonic system which transmitted at 2.5 MHz and received at 5.0 MHz. Background-subtracted myocardial videointensity from the mid-anterior and posterior portions of the myocardium was measured off-line at end-systole from high fidelity videotape images. Gray scale software (Tom-Tec Review Station; Louisville, Colo.) which quantitates videointensity (0 to 255 scale) versus time was used to measure the contrast intensity from a region of interest in the anterior and posterior myocardium. Transmit power and time gain compensation settings on the ultrasound system were kept constant for each individual dog for all comparisons. Visual grading of myocardial contrast enhancement in both the anterior and posterior myocardium was determined by using a scale of 0=no contrast enhancement, 1+=mild contrast enhancement, 2+=bright contrast enhancement. Interobserver variability on these measurements was determined by comparing myocardial contrast intensity measured by two independent experienced observers on 12 of these measurements in three dogs. All comparisons between conventional and transient response imaging were made by using unpaired t testing. The correlation between independent measurements of contrast videointensity between two different reviewers was made with linear regression.

A total of 32 intravenous injections of PESDA were given (16 comparisons). None of these injections using either imaging technique resulted in any change in heart rate, left ventricular systolic or end-diastolic pressure, or change in mean pulmonary artery pressure. Cardiac output also did not change following any injection.

In both first and second harmonic imaging, the myocardial contrast seen with transient response imaging was over three times greater, and was associated with less posterior attenuation. See Table 3 below.

                  TABLE 3                                                          ______________________________________                                         Videointensity of Conventional and TR Imaging                                                            Second                                                       First Harmonic    Harmonic                                                     Imaging           Imaging                                                      Conventional                                                                            Suspended                                                                               Conventional                                                                             Suspended                                          (n = 9)  (n = 10) (n = 9)   (n = 8)                                    ______________________________________                                         Anterior wall                                                                            3 ± 4   23 ± 8*                                                                              17 ± 7*                                                                             60 ± 25**                             Posterior wall                                                                           2 ± 3   8 ± 8 9 ± 7                                                                               30 ± 24**                             ______________________________________                                          *p < 0.5 compared to conventional first harmonic imaging (ANOVA); **p <        0.05 compared to all other groups (ANOVA) TRI = transit response imaging 

Thirteen of the comparisons using transient response imaging were using Method 1, and three involved Method 2. The duration of myocardial contrast using Method 1 averaged 3.4±2.0 seconds. When using Method 2, the myocardial contrast was just as high as Method 1, but was demonstrable for the entire injection period. In the dogs where 2.5 MHz and 3.5 MHz frequencies were compared, the transient contrast intensity was significantly higher with the lower frequency transducer (56±11 units for 2.5 MHz versus 26±7 with 3.5 MHz; p=0.004).

The visual grade of myocardial contrast for transient response imaging was 2+ for 14 of 16 injections compared to only three of 16 intravenous injections using conventional imaging. This degree of visual contrast enhancement was evident for both posterior and anterior myocardium. The correlation in myocardial videointensity measurements by the two different reviewers was 0.93 (p<0.0001), with a standard error of 5 units. The two independent reviewers agreed on the degree of visual myocardial contrast in 10 of 12 random comparisons.

A visually apparent anterior and posterior perfusion abnormality during acute left anterior descending ischemia and left circumflex occlusion in two different dogs was detected using transient response imaging. These contrast abnormalities could not be visualized with conventional imaging. 

What is claimed is:
 1. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing said echo contrast agent into an animal by intravenous injection wherein said echo contrast agent comprises microbubbles the internal atmosphere of which contains perfluorocarbon gas; pulsing an ultrasonic transmission signal in repeating intervals so that contrast of the resulting image is enhanced, and detecting said image.
 2. The method of claim 1 wherein said pulsed ultrasonic transmission is for a time period of approximately of 1 second intervals.
 3. The method of claim 1 wherein said ultrasound signal is pulsed in coordination with said animal's cardiac cycle.
 4. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing an echo contrast agent with perfluorocarbon gas filled microbubbles into an animal by intravenous injection, wherein said perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluorobutane, perfluoroethane, and perfluoropropane or a blood insoluble gas with a molecular weight of greater than 100 g/mole; pulsing an ultrasonic transmission signal in repeating intervals so that contrast of the resulting image is enhanced; and detecting said image.
 5. The method of claim 4 wherein said perfluorocarbon gas is perflurobutane.
 6. The method of claim 4 wherein said microbubbles are encapsulated by a filmogenic protein such as human serum albumin.
 7. The method of claim 6 wherein said human serum albumin is diluted with dextrose.
 8. The method of claim 6 wherein said dilution of human serum albumin with dextrose is three to one.
 9. The method of claim 6 wherein said human serum albumin is a 5% by weight solution and said dextrose is a 5% by weight solution.
 10. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing said echo contrast agent into an animal by intravenous injection; suspending ultrasonic transmission so that image contrast is enhanced; resuming ultrasonic transmission wherein said suspension and reinitiation is triggered by a point in the cardiac cycle; and detecting said ultrasonic image.
 11. A method of ultrasonic myocardial renal or hepatic imaging comprising:preparing an echo contrast agent which comprises microbubbles; introducing said echo contrast agent into an animal; pulsing ultrasonic transmission in repeating intervals timed once every cardiac cycle, and thereafter, resuming ultrasonic transmission.
 12. The method of claim 11 wherein said step of suspending ultrasonic transmission is for a period of approximately 1 second.
 13. A method of ultrasonic myocardial, renal, or hepatic imaging comprising:preparing an echo contrast agent which comprises microbubbles by diluting a solution of 5% by weight albumin with 5% by weight dextrose by 3:1 to create a mixture; introducing said echo contrast agent into an animal; pulsing ultrasonic transmission in repeating intervals timed once every cardiac cycle; and thereafter; resuming ultrasonic transmission.
 14. The method of claim 13 further comprising the step of:sonicating said mixture in the presence of a perfluorocarbon gas.
 15. The method of claim 13 wherein said perfluorocarbon gas is selected from the group consisting of perfluoropropane and perfluorobutane.
 16. The method of claim 13 wherein said perfluorocarbon gas is perfluorobutane.
 17. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing said echo contrast agent into an animal by intravenous injection; triggering an ultrasonic transmission signal in repeating intervals so that contrast of the resulting image is enhanced, said ultrasonic transmission occurring at a pre-selected frequency, and detecting said image at an ultrasound frequency which is a harmonic interval of said transmission frequency.
 18. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing said echo contrast agent into an animal by intravenous injection; triggering an ultrasonic transmission signal in repeating intervals so that contrast of the resulting image is enhanced, said ultrasonic transmission occurring at a pre-selected frequency; and detecting said image at an ultrasound frequency which is a second harmonic interval of said transmission frequency.
 19. A method of ultrasonic myocardial, renal, or hepatic imaging which employs an echo contrast agent having microbubbles comprising:introducing an echo contrast agent with gas filled microbubbles into the vascular system of an animal; inhibiting an ultrasonic imaging signal for a period of time which allows the area to be imaged to be infused with said contrast agent; pulsing said ultrasonic imaging signal following said period to break at least some of said microbubbles and visualize said area to be imaged wherein said period is determined in consideration of the blood flow cycle timing; and detecting said image. 